Immunoassays utilizing colored microparticles are widely used to provide simple, sensitive field and laboratory tests for analytes of clinical interest such as drugs, antibodies, and antigens. Such tests have been applied to analytes in urine, serum, and whole blood.
In typical immunoassays that use a particulate moiety as a detectable label, colored microparticles such as gelatin, dyed latex, or colloidal gold are labeled with a material (e.g. antibody) which binds the analyte of interest. The most common assay utilizing microparticle indicators is the lateral flow immunochromatography assay (see, e.g., U.S. Pat. No. 5,120,643, British Patent GB 2204398A, and European patent EP 0323605 B1). In most lateral flow immunochromatography assays, microparticles are dried onto a sample application pad (typically glass fiber) which in turn is affixed to one end of a strip of chromatographic medium such as nitrocellulose. Another material binding to the analyte of interest is affixed to the chromatographic medium at or near the end opposite to the end having the application pad.
The liquid sample to be analyzed is placed on the pad, causing the suspension of the microparticles into the liquid and allowing any analyte in the liquid sample to bind to the analyte-binding material attached to the microparticles. The liquid sample leaves the application pad by diffusion and capillary action and begins to migrate along the nitrocellulose strip carrying the microparticles down the strip along with the liquid. When the liquid containing the suspended microparticles arrives at the region of the chromatographic strip bearing the second binding material, the analyte (if present in the original sample) will form a molecular bridge between the analyte-binding material on the microparticles and the analyte-binding material affixed to the strip, resulting in the immobilization of the microparticles at that point on the strip where the analyte-binding material is affixed. This immobilization of the microparticles results in a visible signal (e.g., a colored band or dot) at this point on the strip. If the analyte is not present in the sample, the microparticles will continue past this location on the chromatographic strip and a visible signal will not be produced.
A significant problem in the implementation of microparticle-based immunochromatography assays is prevention of non-specific binding of the microparticles to each other (agglutination), to the sample application pad, and to the nitrocellulose strip. Such non-specific binding (binding that is not mediated by specific recognition of the analyte and analyte-binding materials) results in false-positive test results.
A number of strategies have been used to prevent this non-specific binding. These strategies include "blocking" of the sample application pad and nitrocellulose strip with materials such as casein, gelatin, serum albumin, polyethylene glycol, polyvinyl alcohol, and a number of ionic and non-ionic detergents. In other approaches the microparticles are dried on the sample application pad in the presence of some of these same "blocking" materials and "chromatographic transport facilitating agents".
Although the relevant literature typically states that samples suitable for microparticle-based immunoassays include "physiological fluids, for example blood, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid amniotic fluid or the like" (see, e.g., Devereaux et al. EP 0 323 605 B1), in practice such assays have only been applied to blood and urine samples. Saliva, and other oral fluid samples, in particular, pose significant problems in these assays because of the low level of some analytes of clinical interest (e.g. antibodies and antigens relevant to infectious disease), and because of the presence of mucopolysaccharides in saliva which amplify and change the character of the nonspecific binding problems of the microparticles. Accordingly, the routine use of the blocking agents cited above has not proven adequate to produce a practical microparticle-based immunoassay which utilizes a sample containing an oral fluid.